Many natural products with anticancer, antibiotic, and immunosuppressant activity are synthesized by non-ribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). In NRPS and PKS biosynthesis, monomer units are incorporated into a growing natural product chain in an assembly line fashion using a covalent thiotemplate mechanism. Because the growing natural product is covalently tethered to the enzyme, incorporation of a new monomer unit into the growing chain can be measured as a mass shift to the biosynthetic proteins by mass spectrometry. Specifically, Fourier-transform mass spectrometry (FTMS) (and an associated suite of custom MS tools) is a high-resolution analytical technique well suited to the study of NRPS and PKS enzymes because of low detection limits and high mass accuracy measurements.
In this work, FTMS-based studies characterized the biosynthesis of numerous NRPS and PKS derived natural products. In Chapter 2, the development of the phosphopantetheinyl (Ppant) ejection assay and its application to the study of natural product halogenation is presented. Additionally, the expansion of the Ppant ejection assay into a high-throughput liquid chromatography (LC)-MS platform is presented and its application to characterization of mycosubtilin biosynthesis is discussed. In Chapter 3, the on-line Ppant ejection assay using LC-MS is applied to the study of keto- and enoyl reductions during the biosynthesis of the natural product bacillaene. Using FTMS and a variety of biochemical assays, the mechanism of the reductions catalyzed by the enzymes PksJ and PksE were established.
Chapters 4 and 5 discuss the evolution of FTMS from a characterization tool into the basis for a natural product discovery platform. A proteomics platform for the discovery of new natural product biosynthetic gene clusters was developed (PrISM, for the Proteomic Investigation of Secondary Metabolism) to complement previously established genomics and metabolomics approaches. Chapter 4 presents the early development of the PrISM method, including the analysis of a variety of separations and chromatographic techniques and proof-of-concept experiments applying PrISM to known natural product producers. Chapter 5 presents PrISM as a full discovery platform applied to environmental isolates of the genera Bacillus and Streptomyces, where new natural products and undiscovered biosynthetic gene clusters were characterized.